In the Name of God. Talat Mokhtari-Azad Director of National Influenza Center

Transcription

1 In the Name of God Overview of influenza laboratory diagnostic technology: advantages and disadvantages of each test available Talat Mokhtari-Azad Director of National Influenza Center Tehran- Iran 1

2 1) Sampling Diagnosis includes: collection of high-quality specimens 2) Transportation rapid transport to the laboratory and appropriate storage 3) Diagnosis in the laboratory 2

3 Case Selection Select cases based on WHO case definition Fever >38 C, cough and sore throat Quality is more important than quantity Suspected cases Symptoms consistent with influenza case definition For Contacts, NO specimen should be taken systematically: Contacts should be followed-up daily for 7 days and checked if symptoms develop. If symptoms developed, the contact is then considered as a suspected case and therefore specimen should be taken. 3 3

4 Timing 4 4

5 Posterior pharyngeal swab (throat swab) Hold the tongue out of the way with a tongue depressor. Have the subject say "aahh" to elevate the uvula. 5

6 How to Store Specimens Store specimens at 4 C before and during transportation within 48 hours beyond 48 hours store specimens at -70 C and transport using liquid nitrogen tank or dry ice Never store in standard freezer keep on ice or in refrigerator Avoid freeze-thaw cycles Better to keep on ice for a week than to have repeat freeze and thaw 6 6

7 Influenza Diagnosis Diagnosis of influenza can be achieved by: Directly - detecting the virus or viral products Indirectly - detecting an immunological response to virus (Ab detection ) 7

8 Direct Diagnosis of Influenza Virus isolation RNA detection Antigen detection 8

9 Virus isolation 9

10 Virus isolation(1) Virus isolation can be done by : 1- Tissue culture MDCK Cell line, Siat MDCK Cell line 2-Embryonated egg 10

11 Virus Isolation(2) A successful virus isolation depends largely on viral viability of the specimen Important variables include: The timing of the specimen collection in relation to the illness. The quality and amount of specimen material obtained The time and conditions of transport to the laboratory. 11

12 Virus Isolation(3) Advantages: Virus isolation has a higher priority because it can be used for: a: vaccine preparation b: Antiviral resistance studies d: future studies 12

13 Virus Isolation in MDCK cell line Disadvantages: Require specialized facilities Require expert personnel relatively expensive prolonged time to isolate Is not sensitive enough to isolates some strains The growth of viruses in cell culture is often associated with minimal CPE and needs to be detected by Hemagglutination, Hemadsorption or IF tests. 13

14 MDCK Cell Culture A. B. A. non Infected MDCK B. Influenza Infected MDCK 14

15 Hemagglutination test( HAT) RBCs of different species can be used for haemagglutination Test. Avian: turkey or chicken Mammalian: guinea pig or human type O Rhesus Monkey, Horse RBC(H5) Problem: a) The sensitivity of RBCs to agglutinate virus is Changing due to antigenic changes of HA b) Elution of virus from RBCs due to activity of NA Could result in false negative in HAT 15

16 Haemagglutination Assay HA titre Slide from Vicky Gregory 16

17 Haemadsorption Haemagglutinin RBC + haemadsorption Cell monolayer Slide from Vicky Gregory 17

18 Hemadsorption test Slide from Simin Abbasi 18

19 Detection of Antigen by IF Influenza A IF Slide from CDC 19

20 Isolation in embryonated egg(4) Advantages: Easier and faster than tissue culture Virus isolates are suitable for vaccine production Disadvantage: Obtaining embryonated egg (SPF) is not an easy task Some of the subtypes such as (H3N2)is not easily isolated 20

21 The development of monoclonal antibodies in the 1970s increased Rapid viral diagnosis during the 1980s as monoclonal antibody-based assays to detect a wide variety of viral antigens directly in clinical specimens. The introduction of molecular techniques into the diagnostic virology laboratory accelerated with the development of PCR, in 1985, and further with the development of real-time PCR methodology in the late 1990s. 21

22 RNA Detection 22

23 The most common variations of standard PCR used in influenza diagnosis are: Conventional PCR (RT-PCR) Nested PCR (n-pcr) Multiplex PCR (m-pcr) Real-time PCR 23

24 Mulitplex and Nested PCR Multiplex PCR More than one target sequence can be amplified by including more than one pair of primers in the reaction. Nested PCR PCR is performed on the primary PCR product using a new set of internal primers For increased sensitivity 24

25 Nested PCR The advantages of n-pcr are: Its increased specificity (specific binding of 2nd primer pair). Increased sensitivity (2nd round of PCR amplification) n-pcr is used to detect organisms present in low copy Numbers 25

26 Disadvantages of n-pcr n-pcr takes too long Risk of contamination is high 26

27 Multiplex PCR M-PCR is a rapid method of detecting multiple targets in a single reaction. Major advantage is the reduction in test processing time 27

28 Multiplex PCR for Typing 28

29 Multiplex PCR for Subtyping 29

30 Real time PCR Advantages: contamination is decreased, because the system is closed. Rapid cycling times (1 hour). Very sensitive Of great importance, quantitation of PCR targets. 30

31 Conclusion Most of the labs prefer to do PCR for virus detection but PCR is an easy test however when a problem occurs it takes a long time to solve the problem. PCR is expensive Sensitivity is high Decrease the time The lack of requirement for virus viability Reduce virus isolation 31

32 Antigen Detection Source: CDC,USA

33 Antigen Detection Antigen detection methods can provide diagnostic information within a few hours of the receipt of the specimen in the laboratory. The lack of requirement for virus viability is important advantage compared with viral culture, especially when specimen transport time is prolonged. Methods used for viral antigen detection include FA staining, immunoperoxidase staining, and enzyme immunoassay, 33

34 Immunofluoroscence test The main advantage: Direct detection of Ag in patient specimen within the few hours Decrease the time The lack of requirement for virus viability The main disadvantage: Equipment Experienced personnel Require M-Ab Lack of virus 34

35 Near Patient Test Any investigation carried out in a clinical setting or the patient s home for which the result is available without reference to a laboratory and perhaps rapidly enough to affect immediate patient management Hobbs..British Medical Journal

36 Slide from M. Zambon 36

37 Rapid diagnostic tests (1) Advantages: Speed Unskilled Early warning Disadvantages: Expensive Reduce virus isolation Sensitivity/specificity is low Aid management/infection control 37

38 Indirect Method 38

39 Indirect Method Antibody detection/ Serology Hemagglutination Inhibition test (HIT) Neutralization Test (NT) Single Radial Hemolysis(SRH) Plaque Reduction Test (PRT) 39

40 Haemagglutination Inhibition Assay (HAI) 40

41 Serologic Assays (1) Binding Assays: directly measure binding of antibodies to viral antigens. Include HI,EIA, radioimmunoassay (RIA), and the indirect immunofluorescent antibody assay (IFA). Immunobinding Assays: The western blot 41

42 Haemagglutination Inhibition Assay Serological diagnosis (2) Shows presence or absence of antibody against influenza in human sera Antigen characterization HAI can distinguish between Type e.g. A & B Subtype e.g. H1 & H3 42

43 Haemagglutination Inhibition Assay (3) WHO Influenza Kit is used to identify the following antigens and antibodies (to these viruses) in sera: A (H3) A (H1) B (usually more than one lineage) 43

44 Haemagglutination Inhibition Assay Common problems (4) 1- Non-specific inhibitors present in antisera Appears as false positive which could lead to incorrect typing or subtyping 2- Non-specific agglutinins present in sera ( nonspecifically bind RBC) could result in false negative in HIT, especially when HI titers are low 3-Elution of virus from RBCs Could result in false positive in HIT 44

45 Non-specific inhibitors in sera (5) Cross-reaction of virus with non-specific antisera in HI Appears as false positive which could lead to incorrect typing or subtyping Non-specific inhibitors can be removed by RDE treatment 45

46 Non- specific agglutinins in sera (6) Agglutinating proteins present in the sera non-specifically bind to RBCs Identified by agglutination in control sera Could appear as false negative in HI, especially when HI titers are low Non-specific agglutinins can be removed by adsorption of antisera to RBCs 46

47 Haemagglutination Inhibition Assay Elution of RBCs (7) RBCs elute from the HA protein and reduces agglutination (due to NA) This can be overcome by reading results as soon as normal RBCs have settled to a compact button Incubation of plates at 4 o C also slows down elution 47

48 Haemagglutination Assay Avian RBCs (8) Turkey RBCs are preferred to chicken Currently circulating human influenza, particularly H3s, bind better to turkey RBCs than chicken RBCs Chicken RBCs are more readily available Some recently circulating human influenza viruses, particularly H3, bind better with guinea pig RBCs than avian RBCs 48

49 Plaque Assay Slide from Simin Abbasi 49

50 Plaque Reduction test Golden test for Ab detection specially for H5 Golden test for Ab detection after vaccination Golden test for drug resistancy Takes time requirement viable virus Experienced staff 50

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54 Future Concern If a novel virus with the transmissibility of H1N1 and even a fraction of the virulence of H5N1 emerged, the consequences would be devastating. 54

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